Slit-Robo signaling is 1 of the main axon guidance signaling pathways that act to shape the circuitry of the nervous system. Recent work shows the critical role of Slit-Robo signaling in the development of the visual system and spinal cord in model organisms, and the corticospinal tract in humans. However, only a few effector genes have been implicated in this signaling pathway, and none of these effector genes have been studied genetically in vertebrates. In addition, it is poorly understood which structural domains of Robo receptors are critical for their in vivo function. This proposal will use a forward genetic screen searching for interactions (noncomplementation) with the zebrafish astray (robo2) mutant. By screening individual F1 larvae labeled with a GFP transgene, we will be able to screen 75,000 genomes in 3 years. A pilot screen of 5700 genomes has already yielded 12 putative mutants. This screen will (1) identify mutants in genes in the Slit-Robo pathway, then (2) genetically map these mutants and (3) conduct a preliminary phenotypic assessment analyzing multiple axon pathways. We expect to find 3 classes of mutants: (I) new mutant alleles of robo2, which we will identify and sequence; (II) temperature-sensitive robo2 alleles, which we will identify and characterize; and (III) mutations in genes that show strong genetic interactions with robo2, such as slit ligands or downstream effectors, which we will genetically map and analyze phenotypically. The zebrafish system offers unique advantages for studying vertebrate axon guidance, including the ability to trace the trajectories of single axons both in fixed tissue and using timelapse movies; the ability to generate temperature-sensitive alleles in a vertebrate; and the ability to carry out forward-genetic screens. The robo2 alleles isolated in this screen will be valuable for understanding the timing of Robo2 function (temperature-sensitive alleles) and the protein domains necessary for Robo2 signaling (missense or nonsense alleles). The interacting genes isolated will define a set of genes in the Robo2 signaling pathway, and allow us to study their function in vivo.